We have discovered that non-neoplastic rat tissues (uterus, liver, spleen, serum) contain a compound which inhibits 3H-estradiol binding (type II sites) in rat uterine nuclei. Since elevated nuclear levels of type II sites are highly correlated with increased cell proliferation in normal (uterus) and malignant (mouse mammary tumors and human breast cancer) tissues, we feel this inhibitor might be an endogenous anti-proliferative agent. Preliminary studies support this concept in that partially purified preparations of this inhibitor block the growth of normal and malignant cells in culture. Furthermore, estrogen-induced rat mammary tumors are deficient in this inhibitor activity which is consistent with the rapid rate of proliferation in these cell populations. The specific aims of this proposal are to purify this inhibitor to homogeneity and structurally identify the compound(s) by mass spectrometry, nuclear magnetic resonance studies and infra-red spectrometry. In addition we plan to study the interaction of the pure material (purchased; synthesized or prepared from rat liver) with nuclear type II estrogen binding sites (association and dissociation rates; competitive vs. non-competitive inhibition) in normal (rat uterus) and malignant (rat and mouse mammary tumors) tissues. These studies should point out any defects in the ability of nuclear type II sites in malignant tissues to recognize this inhibitor. The effects of pure inhibitor on cell proliferation (cell growth; rate of entry and fraction of cells in S-phase) will be evaluated (rat uterine cells; estrogen-induced rat mammary tumor cells; MCF-7 Human Breast Cancer cells) and these changes correlated to the effects of the inhibitor on nuclear type II estrogen binding sites in these cell populations. Since tumors appear to be deficient in this inhibitor activity, studies will be done to determine whether tumor cells (as described above) or microsomal fractions from estrogen-induced rat mammary tumors will metabolize this inhibitor to an "inactive" form. The results of these experiments may provide a new approach to studies involving the regulation of cell proliferation in normal and neoplastic cells.